Although signal transduction inhibitors occasionally offer clinical benefit for cancer patients, signal flux emanating from oncogenes is often distributed through multiple pathways, potentially underlying the resistance which causes failure of most such inhibitors. Measuring signal flux through multiple pathways, in response to signal transduction inhibitors, may help uncover network inter- actions that contribute to therapeutic resistance and that are not predicted by analyzing pathways in isolation. Protein?protein interactions within signaling pathways are often elucidated by assessing the levels of relevant pathway proteins in model and tumor-derived cell lines and with various genetic and molecular perturbations. Such interactions, and the implied signaling networks, may also be elucidated via quantitative measurements of multiple pathway-related proteins within single cells. At the single-cell level, inhibitory and activating protein?protein relationships, as well as stochastic (single-cell) fluctuations, are revealed. However, most techniques for profiling signaling pathways require large numbers of cells, and bulk measurements have proven insufficient to detect secondary pathways post resistance. Single- cell immunostaining is promising, and some flow cytometry techniques are relevant, yet limited in finding possible pathways due to intracellular multiplexing limitations. We describe quantitative, multiplex assays of intracellular signaling proteins from single cancer cells using a platform called the single-cell barcode chip (SCBC). The SCBC is simple in concept: A single or defined number of cells is isolated within a microchamber that contains a sensitive antibody array specific for the capture and detection of a panel of proteins. The SCBC design permits lysis of each individual trapped cell. Intracellular staining flow cytometry can assay up to 11 phosphoproteins from single cells. Our SCBC can profile a significantly larger panel (up to 90 different phosphoproteins) with ~2500 single cells per chip for a statistically representative analysis of the sample population. This new high multi-plexed single cell phosphoproteomics analysis tool provides an analytical approach for detecting changes in signal coordination by monitoring phosphoproteins, on a much larger scale. This approach may identify actionable alterations in signal coordination that underlie adaptive resistance, which can be suppressed through combination drug therapy, including non- obvious drug combinations. SPECIFIC AIM 1: Develop a robust microchamber array flow cell that can be easily incorporated into larger automated workflow device for analysis of intracellular protein targets. SPECIFIC AIM 2: Double multiplexing capability of high-density barcode SCBC chip by monitoring both intracellular proteins and metabolites simultaneously. Perform single-cell 32-plex measurement for more comprehensive GBM pathway analysis. SPECIFIC AIM 3: Improve consumable to perform ?flow cell? in-cartridge lysis, detection and washing capabilities for automation. Develop fully automated device workflow. SPECIFIC AIM 3b: Demonstrate utility of device in patient clinical trials as a commercial tool.